Polycyclic aromatic hydrocarbons (PAHs), such as three-ringed phenanthrene, four-ringed chrysene, are commonly found as pollutants in soils, estuarine waters and sediments and other aquatic sites. Phenanthrene has been shown to be toxic to marine diatoms (Kusk, 1981), gastropods (Pipe and Moore, 1986), mussels, crustaceans and fish (Black et al., 1983). Inoculation of pure microbial culture to biodegrade recalcitrant organic compounds has had increased interest in the field of biological treatment of polluted waters or wastewater effluents. Several bacteria and cyanobacteria are known to metabolise phenanthrene. White-rot fungi are also known to metabolise phenanthrene besides several other PAHs. White-rot fungi are known to metabolise several xenobiotic compounds due to extracellular production of lignin-degrading enzymes. Due to the importance attached to prevention of environmental pollution, environmental agencies all over the world are imposing strict regulations for mitigation of pollution from industries and human activities. Therefore, search for newer and better sources of organisms for removal or bioremediation of toxic compounds remains a continuous process. F. flavus produces lignin-modifying enzymes such as manganese-dependent peroxidase E.C.1.11.1.7 (MNP), lignin peroxidase, E.C. 1.11.1.7 (LIP) and laccase, E.C. 1.10.3.2 when grown on sugarcane bagasse suspended in distilled water or in conventional media prepared with distilled water or half-strength sea water (Raghukumar et al. 1999a).
The fungus Flavodon flavus belonging to the class Basidiomycetes produces fertile basidiomata in medium containing alpha-cellulose and sometime in malt extract agar medium on a prolonged incubation. Most of the time this fungus is in non-sporulating form, off white to white in color, slimy looking mycelium and can be recognised by crystals deposited around fungal hyphae. Fruiting bodies from culture were identified as Flavodon flavus using the key in Ryvarden Johansen (Ryvarden, L. and I. Johansen, 1980. A preliminary polypore flora of East Africa, Fungiflora, Oslo, Norway). Most of the white-rot fungi do not grow in the presence of synthetic or natural seawater whereas, the strain Flavodon flavus isolated by the applicants from decaying seagrass, grows and produces lignin-degrading enzymes in the presence of half-strength synthetic sea water as well as in distilled water (Chandralata Raghukunar, Trevor, M. D'Souza, Greg Thorn, and C. A. Reddy. 1999. Lignin-modifying enzymes of Flavodon flavus isolated from a coastal marine environment. Applied and Environmental Microbiology, 65: 2103–2111). This was the first report of production of lignin-degrading enzymes in this fungus and also their production in half-strength artificial seawater.
Normally the wastewater disposal includes physical-chemical treatment, waste-minimisation and biological treatment. Most of the biological approaches considered for restoration of PAH-contaminated sites depend on the activity of bacteria. Whereas low-molecular weight-PAH are usually readily degraded, high-molecular weight PAH of five or more rings resist bacterial degradation. White-rot fungi would be expected to have greater access to poorly available substrate, since they secrete extracellular enzymes involved in the oxidation of complex aromatic compounds like lignin. PAHs are recalcitrant, hydrophobic compounds and sorption to biological solids may be a significant mechanism for removal of PAHs from refinery wastewater.
Various white-rot fungi have been tried for biodegradation of PAHs:                (i) A reference may be made to a publication (Sack, U.,Heinz, T. M., Deck, J., Cemglia, C. E., Martens, R., Zadrazil, F. and Fritsche, W. 1997. Comparison of phenanthrene and pyrene degradation by different wood-decaying fungi. Applied and Environmental microbiology 63: 3919–3925) wherein degradation of phenanthrene and pyrene by different wood-decaying fungi is reported. The fungi used were Trametes versicolor, Laetiporus sulphureus, Kuehneromyces mutabilis, Flammulina velutipes and Agrocybe aegerita. A concentration of 50 mgL+1 phenanthrene was used in low nitrogen liquid medium and 3.7% to 15.5% was mineralised in 63 days.        (ii) A reference may be made to a publication (Cuny, P., Faucet, J., Acquaviva, M., Bertrand, J. C. and Gilewicz M. 1999. Enhanced biodegradation of phenanthrene by a marine bacterium in presence of a synthetic surfactant. Letters in Applied Microbiology, 29:242–245), wherein a marine bacterium Sphingomonas sp. is reported to degrade about 85% of 4ppm phenanthrene after 8–9 days in the presence of the surfactant Tween 80. However, no reference is about degradation by heat-killed culture.        (iii) A reference may be made to a publication (Cerniglia, C. E. 1984. Microbial metabolism of polycyclic aromatic hydrocarbons. Advances in Applied Microbiology. 30: 31–71) wherein metabolism of PAHs by bacteria, cyanobacteria, fungi and algae is reviewed. Fungi oxidize PAHs via a cytochrome P-450 monooxygenase to form arene oxides. They form glucurnide and sulfate conjugates of phenolic PAHs and these reactions may be important in the detoxification and elimination of PAHs in the environment.        (iv) A reference may be made to a publication (Sack, U., Hofrichter, M. and Fritsche, W. 1997. Degradation of polycyclic aromatic hydrocarbons by manganese-peroxidase of Nematoloma forwardii. FEMS-Microbiology Letters 152: 227–234) wherein mineralization of phenanthrene was brought about by a lignin-degrading enzyme, manganese-peroxidase of the white-rot fungus Nematoloma frowardii suggesting an important role for white-rot fungi in PAH oxidation. Manganese peroxidase of 1.96 U ml-1 degraded radiolabeled phenanthrene of 10 mg L−1 concentraiton (10 ppm) in a period of 7 days. However, using enzyme on a mass scale is an expensive process.        (v) A reference may be made to a publication (Novotny, C., Erbanova, P., Cajthaml, T., Rothschild, N, Dosoretz, C and Sasek, V. 2000. Irpex lacteus, a white rot fungus applicable to water and soil bioremediation. Applied Microbiology and Biotechnology 54: 850–853) wherein, the white-rot fungus Irpex lacteus was implied in water and soil bioremediation contaminated with PAHs. Live culture and heat-killed control removed 37 and 38% phenanthrene (initial concentration f 25 ppm) spiked in brown soil during 2 months incubation period.        (vi) A reference may be made to a publication (Stringfellow, W. T. and Alvarez Cohen, L. 1999. Evaluating the relationship between the sorption of PAHs to bacterial biomass and biodegradation. Water Research, 33: 2535–2544) wherein sorption of PAHs to biological solids is suggested to be a significant mechanism for removal of PAHs from refinery wastewater.        
Although, the fungus strain used here has the same characteristics as already described in the literature, the novelty lies in its new use in the process of PAH removal under fresh water as well as estuarine conditions by employing live as well as heat-killed fungal biomass.